With the development of Kohler and Milstein's hybridoma technology around 1975, monoclonal antibodies were utilized in the discovery and identification of cell surface molecules. Cell marker analyses are important in the prognosis, classification of state of disease, treatment decisions, and monitoring of therapy. For example, in human immunodeficiency virus (HIV) infection, both the CD4.sup.+ /CD8.sup.+ T-cell ratio and the absolute number of CD4.sup.+ T lymphocytes are important for the reasons noted above. The functions of cell membrane molecules and the consequences of their quantitative changes in several disorders (e.g., septicemia, burns, autoimmune diseases, graft rejection) are better understood because of advances in techniques in molecular biology and in the ability to assess the state of the immune system and to give more accurate prognoses.
Cell membrane markers are usually assayed by flow cytometry using a fluorescence-activated cell sorter (FACS) and a fluorescently labeled monoclonal antibody specific for the cell marker to be assayed. A FACS analysis measures cells as they flow through a flow cytometer in single file, or the best approximation thereof that can be achieved, in a fluid stream. Standardization and reproducibility of tests for clinical application is difficult, especially when measuring quantitative cellular fluorescence intensity, because different flow cytometers differ in sensitivity. In addition, the instrument is expensive (frequently &gt;$200,000) and is labor intensive, requiring highly trained personnel to run the FACS. FACS analysis is also unreliable due to instrument variables and due to the way results are expressed. Usually, the results are expressed in units which require a separate determination of the number of white blood cells per .mu.L of blood and a differential count. This combines the variability of three tests into one clinical result. Consequently, it would not be unusual for the same sample to give results differing by a factor of two in two separate determinations.
Given the foregoing, researchers have endeavored unsuccessfully to reliably determine markers in or on cells using enzyme immunoassays as an alternative to flow cytometry.
Franke et al., Clin. Chem., 40(1): 38-42 (1994) and AIDSLINE, December 1993 reporting on the Int-Conf-AIDS, page 259 (June 16-21, 1991) describe a cell marker ELISA, Capcellia.TM. CD4/CD8, to quantitatively determine CD4/CD8 molecules. The assay is performed in a single step on microtiter plates, specifically, cells are immobilized on the solid phase using pan-T monoclonal antibodies adsorbed on the solid phase surface along with simultaneous labeling of CD4 or CD8 by peroxidase-labeled immunoconjugates. The results are expressed in molar concentrations of CD4 or CD8 molecules calculated from standard curves. The factors used to convert concentrations of CD4 molecules into cells per liter were only relative and were used as guides. Thus, this assay falls woefully short in being an efficient alternative to flow cytometry.
Other approaches include the following:
Baumgarten, J. Immunological Methods, 94: 91-98 (1986) describes the requirements for calibration of a cell ELISA for the quantitation of leukocyte antigens using air-dried and methanol-fixed cells which were attached to microplate wells. The test was standardized by measuring both the specific antigen and the amount of cellular protein in each single sample and was calibrated either with intact cells or isolated plasma membranes prepared from the cells under study.
Hessian et al., J. Immunological Methods, 91: 29-34 (1986) describes a cell-associated enzyme immunoassay as an alternative to FACS analysis by employing immunofiltration methodology and soluble complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase.
U.S. Pat. No. 4,661,466, issued to Schlossman et al. on Apr. 28, 1987, describes the use of a monoclonal antibody to distinguish subsets of cells on the basis of different degrees of reactivity with the monoclonal antibody.
Similarly, U.S. Pat. No. 4,677,061, issued to Rose et al. on Jun. 30, 1987, describes T-cell lymphocyte subset monitoring of immunologic disease. T-cell subsets are monitored for a designated pattern of epitopic sites associated with specific surface membrane proteins where ratios of cells having different patterns are determined by multi-parameter flow cytometric analysis, the ratios being indicative of a probable change in the immunologic disease.
WO 90/04180, published Apr. 19, 1990, describes a method for measuring soluble CD4 antigens to diagnose a state of immune activation.
WO 92/08981, published May 29, 1992, describes the measurement of total leukocyte antigens and the use of such measurements to enumerate cells.
None of these references describes a standardized and reproducible cell enumeration immunoassay which is an efficient alternative to flow cytometry.